NosN, a Radical S-Adenosylmethionine Methylase, Catalyzes Both C1 Transfer and Formation of the Ester Linkage of the Side-Ring System during the Biosynthesis of Nosiheptide

Abstract

Nosiheptide, a member of the e series of macrocyclic thiopeptide natural products, contains a side-ring system composed of a 3,4-dimethylindolic acid (DMIA) moiety connected to Glu6 and Cys8 of the thiopeptide back-bone via ester and thioester linkages, respectively. Herein, we show that NosN, a predicted class C radical S-adenosylmethionine (SAM) methylase, catalyzes both the transfer of a C1 unit from SAM to 3-methylindolic acid linked to Cys8 of a synthetic substrate surrogate as well as the formation of the ester linkage between Glu6 and the nascent C4 methylene moiety of DMIA. In contrast to previous studies that indicated that 5′-methylthioadenosine is the immediate methyl donor in the reaction, in our studies SAM itself plays this role, giving rise to S-adenosylhomocysteine as a co-product of the reaction.

Enhanced Solubilization of Class B Radical S-Adenosylmethionine Methylases by Improved Cobalamin Uptake in Escherichia coli

“…Class C enzymes uniquely methylate, methenylate, or cyclopropanate sp 2 -hydridized carbon centers that are typically found in complex natural products. 28–30 Class D RS methylases are the least characterized. Unlike class A, B, and C enzymes, which transfer a C1 unit from SAM, class D enzymes employ an alternative source of the C1 unit, hypothesized to be a derivative of methylenetetrahydrofolate.…”

Using Peptide Mimics to Study the Biosynthesis of the Side-Ring System of Nosiheptide

“…The plasmid is digested with Nhe I and Xho I, and the fragment containing the nosN gene is ligated into a pET28a expression plasmid digested with the same restriction enzymes. The resulting construct encodes NosN with an N-terminal hexahistidine (His 6 ) tag linked by a thrombin cleavage site for removal of the tag (LaMattina et al, 2017). The pET-based cloning system, coupled with the strong T7 promoter, allows for facile cloning and robust overproduction of proteins (Panavas, Sanders, & Butt, 2009).…”

“…NosN has been stated to produce 5′-deoxyadenosine (5′-dA) and either S-adenosylhomocysteine (SAH) or thioadenosine (tA), depending on whether S-adenosylmethionine (SAM) or methylthioadenosine (MTA) acts as a methyl donor, respectively (Ding, Li, et al, 2017; LaMattina et al, 2017). SAM typically degrades to MTA during long periods of storage, which is heavily dependent on the pH of the storage solution and the temperature.…”

“…The substrate for NosN is more difficult to quantify due to a lack of synthetic protocols to make a peptide mimic that contains the intact side-ring product. One alternative is to use MIA-SNAC as a sub-strate analog for NosN assays and quench with sodium hydroxide to form a hydroxymethyl adduct of MIA (LaMattina et al, 2017). However, such methods result in a mixture of products formed due to a lack of specificity with the poor substrate, making the quantification less meaningful.…”

“…Previous studies of NosN showed that MIA containing an N -acetylcysteamine group (MIA-SNAC) is recognized as a substrate to form DMIA. However, considering the lack of additional gene products that could transform DMIA into a closed side-ring system, we posited that DMIA is formed as a side product when NosN cannot complete its catalytic cycle (Ding, Li, et al, 2017; Ding, Wu, et al, 2017; LaMattina et al, 2017). Only when using substrate analogs that better mimic the predicted physiological one could we probe the full reaction catalyzed by NosN.…”